Downloaded from Consensuspathdb Collection We Defined High-Confidence MTHFD2-Interacting Pro- (

Koufaris and Nilsson Cancer & Metabolism (2018) 6:12 https://doi.org/10.1186/s40170-018-0185-4 RESEARCH Open Access Protein interaction and functional data indicate MTHFD2 involvement in RNA processing and translation Costas Koufaris1,2,3 and Roland Nilsson1,2,3* Abstract Background: The folate-coupled metabolic enzyme MTHFD2 is overexpressed in many tumor types and required for cancer cell proliferation, and is therefore of interest as a potential cancer therapeutic target. However, recent evidence suggests that MTHFD2 has a non-enzymatic function which may underlie the dependence of cancer cells on this protein. Understanding this non-enzymatic function is important for optimal targeting of MTHFD2 in cancer. Methods: To identify potential non-enzymatic functions of MTHFD2, we defined its interacting proteins using co-immunoprecipitation and mass spectrometry and integrated this information with large-scale co-expression analysis, protein dynamics, and gene expression response to MTHFD2 knockdown. Results: We found that MTHFD2 physically interacts with a set of nuclear proteins involved in RNA metabolism and translation, including components of the small ribosomal subunit and multiple members of the RNA-processing hnRNP family. Interacting proteins were also in general co-expressed with MTHFD2 in experiments that stimulate or repress proliferation, suggesting a close functional relationship. Also, unlike other folate one-carbon enzymes, the MTHFD2 protein has a short half-life and responds rapidly to serum. Finally, shRNA against MTHFD2 depletes several of its interactors and yields an overall transcriptional response similar to targeted inhibition of certain ribosomal subunits. Conclusions: Taken together, our findings suggest a novel function of MTHFD2 in RNA metabolism and translation. Keywords: Interactome, Moonlighting, Heat shock proteins, RNA, Non-metabolic Background unclear, but may be due to the mitochondrial NADH/ Altered metabolism is a hallmark of cancer cells that NAD ratio favoring flux in the oxidative direction [8], or facilitates their growth and survival [1]. In particular, me- related to the associated reduction of NAD and NADP tabolism of folate-coupled one-carbon groups is repro- [7]. In the nucleus, the formate produced by the mito- grammed in a wide variety of cancers [2–4]. One-carbon chondrial pathway supports synthesis of dTMP during metabolism is localized into cytosolic, mitochondrial, and DNA replication. nuclear compartments (Fig. 1) and is required for the syn- Within mitochondrial folate one-carbon metabolism, thesis of purines, dTMP, and remethylation of homocyst- the enzyme MTHFD2 has attracted considerable attention eine to methionine. Extensive evidence now supports that as a potential target for cancer therapeutics [4, 9, 10], mo- proliferating cancer cells primarily rely on the mitochon- tivated by a favorable expression profile with high expres- drial catabolism of serine via SHMT2 as their main source sion in various human tumor types [4]butlowor of one-carbon groups [5–7], rather than the correspond- undetectable levels in most adult tissues. The normal ing cytosolic enzymes. The reason for this preference is function of MTHFD2 may be in embryogenesis, since the enzyme has long been known to be highly expressed and * Correspondence: [email protected] essential during embryonic development [11], and a par- 1Cardiovascular Medicine Unit, Department of Medicine, Karolinska Institutet, alogous mitochondrial enzyme, MTHFD2L, is expressed SE-171 76 Stockholm, Sweden 2Division of Cardiovascular Medicine, Karolinska University Hospital, SE-171 76 in normal adult tissues [12]. Expression of MTHFD2 in Stockholm, Sweden tumors also correlates with poor disease outcome in Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Koufaris and Nilsson Cancer & Metabolism (2018) 6:12 Page 2 of 14 nucleus cytosol mitochondrion MTHFD2? serine serine serine SHMT1/2 SHMT1 SHMT2 glycine glycine glycine TYMS dtmp ch2-thf ch2-thf ch2-thf purines MTHFD2 MTHFD1 MTHFD1 nad(p)h cho-thf cho-thf cho-thf ALDH1L2 MTHFD1 MTHFD1 MTHFD1L nadph formate formate formate co2 Fig. 1 Schematic diagram of enzymes, metabolites, and compartmentalization of folate one-carbon metabolism. One-carbon metabolism enzymes are now known to be present and active in three distinct compartments, the nucleus, mitochondria, and cytosol, linked by the flow of formate between them. The function of MTHFD2 in the mitochondria is well understood, although its function in the nucleus is not known breast cancer [13], liver cancer [14], and acute myeloid co-expression pattern and the transcriptional responses leukemia (AML) [10]. Knockdown of MTHFD2 can in- to knockdown. Taken together, our results suggest a pre- hibit cancer cell proliferation in vitro [4, 10, 15] and re- viously unrecognized role for MTHFD2 in RNA metab- duces (but does not abolish) tumor growth in vivo [7, 10]. olism and translation. This combination of specific expression in transformed cells and severe cell phenotypes upon knockdown render Methods MTHFD2 a compelling potential drug target. Cell culture Nevertheless, several observations suggest that the HCT-116 cell lines were obtained from the National − − known metabolic function of MTHFD2 may not be the Cancer Institute. The MTHFD2 CRISPR / knockout sole explanation for its prominent role in cancer. First, (D2-KO) and parental wildtype (WT) HCT-116 cells supplementing cultures with formate, the primary product were obtained from the lab of Dr. Rabinowitz [7]. Cells of the mitochondrial pathway (Fig. 1), fails to rescue were grown in RPMI-1640 medium supplemented with MTHFD2 knockdown cancer cells [4, 10], indicating that 5% FBS (Life technologies) and Penicillin/Streptomycin another function of the MTHFD2 protein might be neces- (Gibco) and maintained at 37 °C and 5% CO2. sary for growth. Second, cells can rapidly adapt to loss of For serum starvation experiments, 200,000 cells were MTHFD2 by shifting to the cytosolic one-carbon pathway plated in six-well plates. The next day, the medium was [7], suggesting a degree of redundancy where flux through removed, the cells were washed with PBS and then the mitochondrial pathway is not strictly essential. Third, serum-free RPMI-1640 medium was added. Cyclohexa- knockdown of the SHMT2 enzyme, which should also mide was obtained from Sigma Aldrich (C4859). For the block the mitochondrial pathway, does not cause cell serum re-stimulation experiments, after 48 h of serum death unless glycine is removed [2, 16]. Moreover, the starvation, wells were washed with PBS and then fresh MTHFD2 protein was recently found to be present in the RPMI-1640 containing 20% FBS was added to the cells. nucleus at sites of newly synthesized DNA, and over-expression of catalytically inactive MTHFD2 drives Real-time PCR cell proliferation [17], suggestive of a role in signaling/ RNA was isolated from cells using the Qiagen RNeasy mini regulation rather than metabolism. Knockdown of kit and quantified using a Nanodrop ND-1000 spectropho- MTHFD2 also affects invasion and migration in vitro [14, tometer. The RNA was reverse transcribed to cDNA using 18], although no obvious mechanistic link exists between the VILO cDNA synthesis kit (Invitrogen). The qPCR one-carbon metabolism and these phenotypes. reactions were performed in triplicate on a StepOne Given these observations, it is becoming clear that the Real-Time PCR machine (Thermo Fisher Scientific) using effective targeting of the MTHFD2 protein requires a the Fast SYBR Green master mix (Thermo Fisher better understanding of its role in cancer cells beyond Scientific). Oligos were ordered from Sigma Aldrich. Primer metabolism. Although chemical inhibitors of the sequences are listed in Additional file 1. Relative amounts MTHFD enzymatic activity have been reported [19, 20], of mRNA were calculated using the ΔΔCT method normal- different strategies may be required if a non-enzymatic ized to RPLPO mRNA as a reference. function of the MTHFD2 enzyme is critical for cancer cells. For these reasons, we performed an initial investi- Immunoblotting gation of the possible non-metabolic functions of Total protein was isolated from cells using RIPA buffer MTHFD2 by mapping the protein’s interacting partners, (Thermo Fisher Scientific), with the addition of × 100 Koufaris and Nilsson Cancer & Metabolism (2018) 6:12 Page 3 of 14 Halt protease inhibitors (Thermo Fisher Scientific) just Q Exactive HF Orbitrap mass spectrometer, Thermo prior to the extraction. Protein from nuclear and cyto- Scientific). The peptides were separated on an Easy-C18 solic cellular compartments were fragmented as de- column, 50 cm (Thermo Fisher Scientific) at 55 °C in a scribed previously [17]. Protein levels were quantified by 120-min gradient at a flow rate of 300 nL/min. The lin- the BCA assay (Pierce). Equal amounts of protein (10– ear gradient was from 5 to 26% of buffer B (98% aceto- 20 μg) were loaded per lane of 10% SDS-PAGE gel. The nitrile/0.1% formic acid) in 115 min and to 95% of following primary antibodies were incubated overnight: buffer B in 5 min. anti-MTHFD2 (Proteintech 12270-1-AP); anti-MTHFD1 We used data-dependent acquisition with a survey scan (Proteintech 6113-1-AP); anti-MTHFD1L (Proteintech range of 300 to 1650 m/z, at a resolution of 120,000 m/z 10794-1-AP); Lamin A/C (Thermo Fisher Scien- and selected up to 16 most abundant features with a tific MA3-1000); HSP60 (Abcam ab46798); Tubulin charge state ≥2 for HCD fragmentation at a normalized (Abcam ab6046); and COX IV (Abcam 33985).

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